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In JoVE (1)

Other Publications (20)

Articles by Stefán R. Jónsson in JoVE

 JoVE Immunology and Infection

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

1Institute for Experimental Pathology, University of Iceland


JoVE 3483

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Other articles by Stefán R. Jónsson on PubMed

Vascular Dysfunction in Aging: Potential Effects of Resveratrol, an Anti-inflammatory Phytoestrogen

Epidemiological studies demonstrated that even in the absence of other risk factors (e.g. diabetes, hypertension, hyperhomocysteinemia, hypercholesterolemia), advanced age itself significantly increases cardiovascular morbidity by enhancing vascular oxidative stress and inflammation. Because the population in the Western world is rapidly aging, there is a substantial need for pharmacological interventions that delay the functional decline of the cardiovascular system. Resveratrol is an atoxic phytoestrogen found in more than 70 plants including grapevine and berries. Recent data suggest that nutritional intake of resveratrol and other polyphenol compounds may contribute to the "French paradox", the unexpectedly low cardiovascular morbidity in the Mediterranean population. There is increasing evidence that resveratrol exerts multifaceted anti-oxidant and/or anti-inflammatory effects in various disease models. Importantly, resveratrol was reported to slow aging and increase lifespan in simple organisms and has been suggested as a potential calorie restriction mimetic. Resveratrol has also been reported to activate NAD-dependent histone deacetylases (sirtuins), which may contribute to its anti-aging effects. This review focuses on the role of oxidative stress and inflammation in cardiovascular dysfunction in aging, and on emerging anti-aging therapeutic strategies offered by resveratrol and other polyphenol compounds.

Testing and Improving Experimental Parameters for the Use of Low Molecular Weight Targets in Array-CGH Experiments

Array-comparative genomic hybridization (CGH) has evolved as a useful technique for the detection and characterization of deletions, and, to a lesser extent, of duplications. The resolution of the technique is dictated by the genomic distance between targets spotted on the microarray, and by the targets' sizes. The use of region-specific, high-resolution microarrays is a specific goal when studying regions that are prone to rearrangements, such as those involved in deletion syndromes. The aim of the present study was to evaluate the best experimental conditions to be used for array-CGH analysis using low molecular weight (LMW) targets. The parameters tested were: the target concentration, the way LMW targets are prepared (either as linearized plasmids or as purified PCR products), and the way the targets are attached to the array-CGH slide (in a random fashion on amino-silane coated slides, or by one amino-modified end on epoxysilane-coated slides). As a test case, we constructed a microarray harboring LMW targets located in the CREBBP gene, mutations of which cause the Rubinstein-Taybi syndrome (RTS). From 10 to 15% of RTS patients have a CREBBP deletion. We showed that aminosilane- and epoxysilane-coated slides were equally efficient with targets above 1,000 bp in size. On the other hand, with the smallest targets, especially those below 500 bp, epoxysilane-coated slides were superior to aminosilane-coated slides, which did not allow deletion detection. Use of the high resolution array allowed us to map intragenic breakpoints with precision and to identify a very small deletion and a duplication that were not detected by the currently available techniques for finding CREBBP deletions.

Resveratrol Inhibits Aggregation of Platelets from High-risk Cardiac Patients with Aspirin Resistance

Up to 20% of serious vascular events in high-risk vascular patients is attributable to a failure of aspirin (ASA) to suppress platelet aggregation. Resveratrol is a cardioprotective phytoestrogen that can inhibit platelet aggregation in animal models. We hypothesized that resveratrol can also inhibit aggregation of platelets from ASA-resistant (ASA-R) patients. Thus, platelet-rich plasma was isolated from ASA-sensitive (ASA-S) and ASA-R patients (aspirin resistance was defined as higher-than-expected aggregation to collagen and epinephrine [>/=40%] after oral treatment with 100 mg/d ASA). Aggregation to adenosine diphosphate (ADP; 5 and 10 mumol/L), collagen (2 mug/mL), and epinephrine (10 mumol/L) in the absence and presence of resveratrol (10 mol/L) was measured by optical aggregometry. Maximal aggregation to 5 mumol/L ADP was only slightly affected by resveratrol. Similar results were obtained using 10 mumol/L ADP. Maximal aggregation of ASA-R platelets to collagen was significantly decreased by resveratrol, whereas resveratrol had only marginal effects in ASA-S platelets. Similar results were obtained with epinephrine as well. Collectively, resveratrol effectively inhibited collagen- and epinephrine-induced aggregation of platelets from ASA-R patients, which may contribute to its cardioprotective effects in high-risk cardiac patients.

Spectrum of CREBBP Gene Dosage Anomalies in Rubinstein-Taybi Syndrome Patients

The Rubinstein-Taybi syndrome (RTS) is a rare autosomal-dominant disease associated with 10-15% of cases with 16p13.3 microdeletions involving the CREB-binding protein gene (CREBBP). We used array-comparative genomic hybridization and Quantitative multiplex fluorescent-PCR (QMF-PCR) to search for dosage anomalies in the 16p13.3 region and the CREBBP gene. We first constructed a microarray covering 2 Mb that carries seven BAC and 34 cosmid clones, as well as 26 low-molecular-weight probes (1000-1500 bp) that are spread along the CREBBP gene. To increase further the resolution inside the CREBBP gene, we used QMF-PCR assays providing a 7 kb resolution. The deletions characterized in this work extended between as little as 3.3 kb and 6.5 Mb. Some deletions were restricted to just a few exons of CREBBP, some deleted either the 5' or the 3' end of the gene plus adjacent genomic segments, others deleted the whole gene away. We also identified a duplication of exon 16. We showed that CREBBP dosage anomalies constitute a common cause of RTS. CREBBP high-resolution gene dosage search is therefore highly recommended for RTS diagnosis. No correlation was found between the type of deletion and the patients' phenotype. All patients had typical RTS, and there was no particular severity associated with certain alterations.

Inhibition of NAD(P)H Oxidase Attenuates Aggregation of Platelets from High-risk Cardiac Patients with Aspirin Resistance

Up to one-third of serious vascular events in high-risk patients is attributable to a failure of aspirin (ASA) to suppress platelet aggregation. We hypothesized that inhibition of NAD(P)H oxidase may inhibit aggregation of platelets from ASA-resistant (ASA-R) patients. Thus, platelet-rich plasma was isolated from ASA-sensitive (ASA-S) and ASA-R patients (aspirin resistance was defined as higher than expected aggregation to collagen and epinephrine [> or = 40%] after chronic oral treatment with 100 mg/day ASA). Aggregation to adenosine diphosphate (ADP) (5 and 10 micromol/l), collagen (2 microg/ml) and epinephrine (10 micromol/l) in the absence and presence of the NAD(P)H oxidase inhibitors: diphenylene iodonium (DPI) (1 micromol/l) and apocynin (3 x 10(-4) mol/l) was measured by optical aggregometry. Maximal aggregation of ASA-R platelets to collagen and epinephrine was significantly decreased by DPI and apocynin, whereas they had no effect in ASA-S platelets. Maximal aggregation to ADP was unaffected by NAD(P)H oxidase inhibition in either group. In ASA-R platelets both NADPH-driven O2(.-) production (lucigenin chemiluminescence assay) and expression of gp91phox and p67phox subunits of the NADPH oxidase (Western blotting) tended to increase. Collectively, inhibition of NAD(P)H oxidase effectively suppressed collagen and epinephrine-induced aggregation of platelets from ASA-R patients, which may represent a novel pharmacological target for cardioprotection in high-risk cardiac patients.

A 580 Kb Microdeletion in 17q21.32 Associated with Mental Retardation, Microcephaly, Cleft Palate, and Cardiac Malformation

We report on a young boy carrying a de novo 580 kb deletion in the 17q21.32 chromosomal band detected by array-CGH. He had multiple malformations including cardiac abnormalities, cleft palate, mental retardation, microcephaly, pronounced metopic suture and other minor facial dysmorphic features. This is the first case reported in the literature with such a small deletion in 17q21.32. This region includes 15 genes.

Genetic Diagnosis of Familial Hypercholesterolemia Using a DNA-array Based Platform

The aim of this study was to validate the Lipochip genetic diagnostic platform by assessing effectiveness, sensitivity, specificity and costs for the identification of patients with familial hypercholesterolemia (FH) in Spain. This platform includes the use of a DNA micro array, the detection of large gene rearrangements and the complete resequencing of the low-density lipoprotein receptor gene.

[Gastroduodenal Ulcer Before and After Helicobacter Pylori]

Before the discovery of Helicobacter pylori, ulcer disease was considered as the result of a conflict between gastric acid and pepsin, on one side, and protection afforded by gastric mucosal barrier, on the other side. The discovery of H. pylori by Marshall and Warren in 1982 overthrew this conception and revealed ulcer disease mainly as an infectious disease. H. pylori eradication with an appropriate triple therapy is now considered as the gold standard treatment of gastroduodenal ulcer. The pathogenic role of H. pylori lies far beyond ulcer disease since H. pylori is looked as involved in nonulcer dyspepsia, nonsteroidal anti-inflammatory drug ulcers, gastric cancer, MALT lymphoma and, eventually, oesophageal adenocarcinoma, and nondigestive diseases as cardiovascular diseases. The pandemic nature of the H. pylori infection, particularly within developing countries, combined with emerging resistances to antibiotics make the development of a vaccine a public health necessity. The relationships between the human host and the bacterium remains mostly unknown, some of which could be beneficial.

2.3 Mb Terminal Deletion in 12p13.33 Associated with Oculoauriculovertebral Spectrum and Evaluation of WNT5B As a Candidate Gene

We describe a patient presenting with developmental delay, patent foramen ovale, moderate short QT interval, and facial dysmorphism including left microtia, preauricular tag and pit, wide left corner of the mouth, and left hemifacial microsomia, fitting with the oculoauriculovertebral spectrum. We identified a de novo 2.3 Mb deletion in the 12p13.33 region that contains eighteen genes. Amongst those, the WNT5B gene stands out as a possible candidate. However, we did not find any mutation of this gene neither in our patient nor in a series of 53 OAVS patients. The CACNA1C gene is interrupted by the centromeric breakpoint of the deletion and its inactivation probably accounts for the short QT interval of the patient. We speculate that the phenotype of our patient may be explained by the combined effect of the loss of several of the genes contained in the deleted chromosomal segment and of the inactivation of CACNA1C.

[Postoperative Complete Abdominal Dehiscence: Risk Factors and Clinical Correlations]

The purpose of this study is to review our clinical experience with abdominal wound dehiscence in the Surgical Department of City Hospital Timisoara. PATIENTS AND METHODS: 19.116 abdominal procedures were performed between January 1992 - March 2009 in our Department and 29 complete dehiscences were identified (0,15%). Significant risk factors in our analysis were intraabdominal infection, wound infection, emergency surgery, malignancies, digestive fistulae, hiperabdominal pressure, sex and age over 65 years. Less significant factors were the abdominal type of incision, the method of wound closure and heart or respiratory diseases. CONCLUSIONS: postoperative complete dehiscence is a constant presence in a surgical department; despite its low frequency, wound dehiscence is associate with a hight mortality and morbidity rate, and increase the costs and hospitalisation periode. Risk factors evaluation and their associations represente an important role in the therapeutic management of the surgical patient.

[Hepatic Hemostasis with Packing in Complex Abdominal Traumatic Lesions: Indications and Postoperative Outcomes]

The purpose of this study is to review our postoperative outcomes with liver packing in complex abdominal trauma. PATIENTS AND METHODS: 76 liver trauma were admitted for operative procedures in the Surgical Department of City Hospital Timisoara between April 1994 - September 2009 and 16 cases were identified in our series as requiring liver packing. In all cases, this method was efficient, with no postoperative bleeding. In the same time, there were specific complications such as bile leak or abdominal collections. CONCLUSIONS: despite a second procedure for packs removal and the possibility for specific complications, liver packing is an efficient method for severe liver trauma or complex abdominal lesions.

Impact of Age on the Relationships of Brown Adipose Tissue with Sex and Adiposity in Humans

Brown adipose tissue (BAT) regulates energy homeostasis and fat mass in mammals and newborns and, most likely, in adult humans. Because BAT activity and BAT mass decline with age in humans, the impact of BAT on adiposity may decrease with aging. In the present study we addressed this hypothesis and further investigated the effect of age on the sex differences in BAT activity and BAT mass.

Haplotype Analyses, Mechanism and Evolution of Common Double Mutants in the Human LDL Receptor Gene

Familial hypercholesterolemia (FH), an autosomal dominant inherited disorder resulting in increased levels of circulating plasma low-density lipoprotein (LDL), tendon xanthomas and premature coronary artery disease (CAD), is caused by defects in the LDL receptor gene (LDLR). Three widespread LDLR alterations not causing FH (c.1061-8T>C, c.2177C>T and c.829G>A) and one mutation (c.12G>A) with narrow geographical distribution and thought to cause disease were investigated. In an attempt to improve knowledge on their origin, spread and possible selective effects, estimations of the ages of these variants (t generations) and haplotype analysis were performed by genotyping 86 healthy individuals and 98 FH patients in Spain for five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C, and c.2232G>A; most patients carried two of these LDLR variants simultaneously. It was found that both the c.1061-8T>C (t = 54) and c.2177C>T alterations (t = 62) arose at about the same time (54 and 62 generations ago, respectively) in the CGCTG haplotype, while the c.12G>A mutation (t = 70) appeared in a CGCCG haplotype carrying an earlier c.829G>A alteration (t = 83). The estimated ages of selectively neutral alterations could explain their distribution by migrations. The origin of the c.12G>A mutation could be in the Iberian Peninsula; despite its estimated age, a low selective pressure could explain its conservation in Spain from where it could have spread to China and Mexico, since the sixteenth century through the Spanish/Portuguese colonial expeditions.

The Influence of Weather and Geographical Conditions on Flight Dynamics of WCR Adults

Since the appearance of western corn rootworm (Diabrotica virgifera virgifera Le Conte) (Coleoptera: Chrysometidae) in Romania, many researchers have been made, in more or less success. In this study we try to clarify the ecology of the species in order to predict its evolution and its potential area of distribution. In Romania, this species was signalled for the first time in 1996, in western part of country neighbouring with Hungary. This region is an important area for maize production for seed and silage. Together, the climatic conditions, altitude and their influence on species behaviour and distribution have become more apparent. Their habitat and survival strategies are strongly dependent on local weather patents and altitude. In order to investigate the potential impact of weather and altitude on pest populations, a clear understanding of the nature and characterization of pest is required. In general, most pest species are influenced by warm, rainfall and altitude. Taking into consideration our data from the years 2008 and 2009, we can emphasize a very serious influence of air temperature, rainfall and altitude on WCR flight dynamics in adults. Dry and warm conditions generally lead to increasing of insects' number. Our data shown positive correlations between air temperature (daily mean) and adults number captured on pheromone traps (daily mean), but the there are limits from which these become negative. The same trend was recorded in previous research period (2004-2006). The rainfall is an important factor that influences adults' dynamics in maize fields. Excessive rainfall leads to adults' number decreasing. Our daily observations showed a decreasing number of beetles while rainfall increases. Regarding the altitude, we observed a decreasing number of WCR adults at once the attitude increase. For capture of adults we used pheromone traps, in 3 replications (T1, T2 and T3) at difference altitude where maize was grown. Significant relationship of WCR flight dynamic with weather and geographical conditions were found. Activities were carried out under the PN-II-ID-PCE-2007-1/RO project.

The Effectivenes of Trichotim in Control of Cydia Pomonella L. in Western Romania

In the last years a significant attack increasing of codling moth was observed in the western part of Romania. In order to reduce the population of this pests a bioproduct was used named Trichotim based on entomophagous insect Trichogramma sp. Trichotim is a Romanian product certified in year 2000 with the purpose to control the pests present in various cultures such us cabbage, vineyards and fruits plantations. The study was expanded over two years (2008 and 2009), in a super intensive apple orchard of Didactical Experimental Station part of University of Agricultural Sciences Timisoara. In order to control the codling moth, the product Trichotim was used three times correlated to pest biology (two releasing of Trichogramma wasps in the time of first generation and one release in the time of second generation of the pest), releasing 300.000 wasps/ha in total, where 250.000 wasps in first generation time of the codling moth respectively 50.000 wasps in the time of second generation of the pest. The releasing of entomophagous species Trichogramma succeeded at two days after maximal flight values of Cydia pomonella L. The flight curves was realized based on pheromonal traps ATRAPOM placed in tree canopy in stage BBCH 69 (end of flowering, all petals fallen). In order to assess the efficacy of Trichotim the observation was conducted 14 days before fruit harvesting. In the plots where 300.000 wasps were released, the fruits were attacked in percent of 12,33% (efficacy 87,67%) in year 2008 and respectively 14,66% (efficacy 85,33%) in 2009. In untreated plots (control) the fruits were attacked in percent of 23% in 2008 respectively 27,33% in 2009.

Weed Control in Conventional and Transgenic Maize with Resistance to Glyphosate

The researches were conducted in order to observe the behaviour of conventional and glyphosate resistant transgenic maize to different weed control methods. In this paper, the obtained results are presented. The study was conducted in experimental years 2008-2009 in the frame of Didactical Station USAMVB Timisoara. In order to conduct this study, 4 variants cultivated with conventional maize DKC 5143 and 8 variants cultivated with transgenic maize DKC-MON88017 with resistance against Diabrotica virgifera virgifera and to glyphosate. The efficacy of weed control methods was assessed, as well as the herbicide selectivity to cultivated maize hybrid. The weed coverage degree in control plot (V2) was 304 weeds/sqm in the first year and 465 weeds/sqm in the second year. In the variants cultivated with transgenic maize the control was up to 90% much more than control percent achieved in conventional variants. Although, in order to achieve an efficient control (higher than 95%), even to transgenic maize, two glyphosate sequential treatments has to be done. The yield results were positive correlated to the different control methods. However those were affected by climatic conditions recorded in experimental years.

Functional Analysis of LDLR Promoter and 5' UTR Mutations in Subjects with Clinical Diagnosis of Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is a dominant disorder due to mutations in the LDLR gene. Several mutations in the LDLR promoter are associated with FH. Screening of 3,705 Spanish FH patients identified 10 variants in the promoter and 5' UTR. Here, we analyse the functionality of six newly identified LDLR variants. Mutations located in the LDLR promoter regulatory elements R2 and R3 (c.-155_-150delACCCCinsTTCTGCAAACTCCTCCC, c.-136C>G, c.-140C>G, and c.-140C>T) resulted in 6 to 15% residual activity in reporter expression experiments and changes in nuclear protein binding affinity compared to wild type. No reduction was observed when cells were transfected with c.-208T, c.-88A, and c.-36G mutant fragments. Our results indicate that mutations localized in R2 and R3 are associated with hypercholesterolemia, whereas mutations outside the LDLR response elements are not a cause of FH. This data emphasizes the importance of functional analysis of variants in the LDLR promoter to determine their association with the FH phenotype.

New Contributions to the Study of Common Double Mutants in the Human LDL Receptor Gene

Variations in the gene encoding the low-density lipoprotein receptor (LDLR) can cause familial hypercholesterolemia (FH), one of the most common inherited metabolic disorders in humans. The functional effects of the p.Gln92Glu and p.Asn564His alterations are predicted as benign, but the c.313 + 1G>C and p.Lys799_Phe801del changes are believed to cause disease. Although p.Gln92Glu and c.313 + 1G>C have been observed only in Spain, p.Asn564His and p.Lys799_Phe801del are widespread in Western Europe. In order to estimate the ages (t generations) of these four variants of the gene, to determine their possible origin and to consider the influence of age and selective pressure on their spread, we analyzed 86 healthy individuals and 126 FH patients in Spain. Most of the FH patients investigated carried two of these four LDLR variants simultaneously, while only one patient carried three of them simultaneously. Haplotype analyses were based on five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C and c.2232G>A. The results suggest that p.Gln92Glu and c.313 + 1G>C arose at about the same time (99 and 103 generations ago, respectively) in the CACTG haplotype and that p.Asn564His and p.Lys799_Phe801del appeared in the CGCCG haplotype and might be slightly more recent variations (92 and 95 generations ago, respectively). Low selective pressures could explain the maintenance of these variants in spite of their ages. The origin of p.Gln92Glu and c.313 + 1G>C appears to be in Spain whereas p.Asn564His and p.Lys799_Phe801del could have been introduced in Spain by Celtic migrations in the seventh to fifth centuries BC.

Molecular Characterization of Familial Hypercholesterolemia in Spain

Familial hypercholesterolemia (FH), characterized by isolated elevation of plasmatic low-density lipoprotein (LDL) cholesterol and premature coronary heart disease (CHD), is associated with mutations in three major genes: LDL receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin 9 (PCSK9). We have analyzed 5430 Spanish index cases and 2223 relatives since 2004 with LIPOchip(®) genetic diagnostic platform, a microarray for the detection of Spanish common mutations in these three genes, including copy number variation (CNV) in LDLR, followed by sequencing analysis of the coding regions of LDLR and exon 26 of APOB, when the result is negative. Samples were received from hospitals of all around Spain. The preferred clinical criterion to diagnose FH was Dutch Lipid Clinic Network (DLCN) score. Our results show that there is a broad spectrum of mutations in the LDLR gene in Spain since about 400 different mutations were detected, distributed along almost the whole LDLR gene. Mutations in APOB (mainly p.Arg3527Gln) covered 6.5% of positive cases and only one PCSK9 mutation was detected. We found correlation between more severe mutations and the clinical diagnosis but also that 28% of FH patients harboring mutations do not have a definite clinical diagnosis. This study analyzes the mutation spectrum in Spain, remarks the importance of genetic diagnosis of FH patients, as well as the cascade screening, and shows how it is being carried out in Spain.

Functional Characterization of Splicing and Ligand-binding Domain Variants in the LDL Receptor

Familial hypercholesterolemia (FH) is an autosomal dominant disorder mostly caused by mutations in the LDLR gene. Although the detection of functional mutations in the LDLR gene provides an unequivocal diagnosis of the FH condition, there are many variants whose pathogenicity is still unknown. The aims of this study were to set up a rapid method to determine the effect of LDLR mutations, thereby providing an accurate diagnosis of FH, and to functionally characterize six LDLR mutations detected at high frequency by the LIPOchip(®) platform (Progenika Biopharma, Spain) in the Spanish population. LDLR expression and activity were analyzed by one-single-step flow cytometry assay and confocal microscopy. Splicing effects were determined by sequencing reverse transcription polymerase chain reaction products. The analysis of three heterozygous variants with a single point mutation within the low-density lipoprotein binding domain allowed us to classify the c.806G>A variant as nonpathogenic, and c.862G>A and c.895G>A variants as causative of FH. The results obtained for three variants affecting donor splice sites of the LDLR mRNA, c.313+2dupT, c.1186+5G>A, and c.1845+1G>C, demonstrated that these mutations are pathogenic. These results expand our knowledge of mutations responsible for FH, providing an accurate diagnosis and leading to early treatment to reduce the risk of premature cardiovascular events.

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